The anti-cancer drugs used to treat tumours are in most cases applied systemically and spread through the whole body of the patient. The high systemic dose of such drugs required for cancer treatment is combined with unpleasant side-effects for the patient.
In an attempt to circumvent this problem, cancer-prodrugs that have to be metabolized or activated in the body before they become cytotoxic have been used. Unfortunately, human tumours that contain appropriate high levels of the activating enzymes are rare. The main site for activation of prodrugs is the liver and to ensure that a tumour, at a distant site, receives a sufficient dose of the activated drug, the amount of activated prodrug produced in the liver has to be quite high and again this leads to toxic side effects for the patient.
One strategy by which these problems of high systemic concentration of activated drugs could be circumvented would be to provide means for activation of the prodrug directly in or near the site of the tumour. This strategy would require that tumour cells, or cells at the site of a tumour are genetically transformed to produce high amounts of the enzymes required for metabolizing the cancer prodrugs. Retroviral vectors are ideally suited for the stable delivery of genes to cells since the retrovirus is able to integrate the DNA form of its genome into the genome of the host cell and thus all daughter cells of an infected cell will carry the retroviral vector carrying the therapeutic gene. A further advantage is that most retroviruses only infect dividing cells and they are therefore ideal gene delivery vehicles for tumour cells.
Retroviral vectors are the most commonly used gene transfer vehicles for the clinical trials that have been undertaken to date. Most of these trials have, however, taken an ex vivo approach where the patient""s cells have been isolated, modified in culture and then reintroduced into the patient.
The delivery of genes in vivo introduces a variety of new problems. First of all, and above all, safety considerations have to be addressed.
A major concern for eventual in vivo gene therapy, both from a safety stand point and from a purely practical stand point, is the targeting of the expression. It is clear that therapeutic genes carried by vectors should not be indiscriminately expressed in all tissues and cells, but rather only in the requisite target cell. This is especially important when the genes to be transferred are such prodrug activating genes designed to ablate specific tumour cells. Ablation of other, non-target cells would obviously be very undesirable.
The essentially random integration of the proviral form of the retroviral genome into the genome of infected cells has posed a serious ethical problem because such random integration may lead to activation of proto-oncogenes and thus lead to the development of a new cancer. Most researchers would agree that the probability of a replication defective retrovirus, such as all those currently used, integrating into or near a cellular gene involving in controlling cell proliferation is vanishingly small. However, it is generally also assumed that the explosive expansion of a population of replication competent retroviruses from a single infection event, will eventually provide enough integration events to make such a phenotypic integration a very real possibility.
Retroviral vector systems are optimized to minimize the chance of replication competent virus being present. It has however, been well documented that recombination events between components of the retroviral vector system can lead to the generation of potentially pathogenic replication competent virus and a number of generations of vector systems have been constructed to minimize this risk of recombination (Salmons, B. and Gxc3xcnzburg, W. H., Human Gene Therapy, 4(2):129-41 (1993).
Retroviral vector systems consist of two components:
1) The retroviral vector itself is a modified retrovirus (vector plasmid) in which the genes encoding for the viral proteins have been replaced by therapeutic genes. Since the replacement of the genes encoding for the viral proteins effectively cripples the virus it must be rescued by the second component in the system which provides the missing viral proteins to the modified retrovirus.
The second component is:
2) A cell line that produces large quantities of the viral proteins, however lacks the ability to produce replication competent virus. This cell line is known as the packaging cell line and consists of a cell line transfected with one or more plasmids carrying the genes enabling the modified retroviral vector to be packaged.
To generate a recombinant retroviral particle, the retroviral vector is transfected into the packaging cell line. Under these conditions the modified retroviral genome including the inserted therapeutic gene is transcribed from the retroviral vector and packaged into the modified retroviral particles. These recombinant retroviral particles are then used to infect tumour cells during which the vector genome and any cytotoxic gene becomes integrated into the target cell""s DNA. A cell infected with such a recombinant viral particle cannot produce new vector virus since no viral proteins are present in these cells but the DNA of the vector carrying the therapeutic is integrated in the cell""s DNA and can now be expressed in the infected cell.
A number of retroviral vector systems have been previously described that should allow targeting of the carried cytotoxic genes (Salmons, B. and Gxc3xcnzburg, W. H. Human Gene Therapy, 4(2):129-41 (1993)). Most of these approaches involve either limiting the infection event to predefined cell types or using heterologous promoters to direct expression of linked heterologous therapeutic genes to specific tumour cell types. Heterologous promoters are used which should drive expression of linked genes only in the cell type in which this promoter is normally active or/and additionally controllable. These promoters have previously been inserted, in combination with the therapeutic gene, in the body of the retroviral vectors, in place of the gag, pol or env genes.
The retroviral Long Terminal Repeat (LTR) flanking these genes carries the retroviral promoter, which is generally non-specific in that it can drive expression in many different cell types (Majors, J. (1990). in xe2x80x9cRetrovirusesxe2x80x94Strategies of replication (Swanstrom, R. and Vogt, P. K., Eds.): Springer-Verlag, Berlin: 49-92). Promoter interference between the LTR promoter, and heterologous internal promoters, such as the tissue specific promoters, described above, has been reported. Additionally, it is known that retroviral LTR""s harbour strong enhancers that can, either independently, or in conjunction with the retroviral promoter, influence expression of cellular genes near the site of integration of the retrovirus. This mechanism has been shown to contribute to tumourigenicity in animals (van Lohuizen, M. and Berns, A. (1990), Biochim. Biophys. Acta, 1032:213-235). These two observations have encouraged the development of Self-Inactivating-Vectors (SIN) in which retroviral promoters are functionally inactivated in the target cell (WO 94/29437). Further modifications of these vectors include the insertion of promoter gene cassettes within the LTR region to create double copy vectors (WO 89/11539). However, in both these vectors the heterologous promoters inserted either in the body of the vector, or in the LTR region are directly linked to the therapeutic gene.
The previously described SIN vector mentioned above carrying a deleted 3xe2x80x2LTR (WO 94/29437) utilizes in addition a heterologous promoter such as that of Cytomegalovirus (CMV), instead of the retroviral 5xe2x80x2LTR promoter (U3-free 5xe2x80x2LTR) to drive expression of the vector construct in the packaging cell line. A heterologous polyadenylation signal is also included in the 3xe2x80x2LTR (WO 94/29437).
A variety of cytotoxic genes carried by retroviral vectors have already been tested. These genes encode enzymes which convert substances that are pharmacodynamically and toxicologically inert even at high dose-levels but which can be converted in vivo to highly active metabolites (Connors, T. A., Gene Therapy, 2:702-709 (1995)).
In cancer chemotherapy appropriately designed prodrugs have been found to be effective in the treatment of animal tumours possessing high levels of an activating enzyme (Connors, T. and Whisson, M., Nature, 210:866 867 (1966); Cobb, L. et al., Biochemical Pharmacology, 18:1519-1527 (1969)). Clinical results were, however, disappointing since it was found that human cancers that contained appropriately high levels of activating enzymes were rare (Connors, T., Xenobiotica, 16:975-988 (1986)). Viral directed enzyme prodrug therapy (VDEPT) and the more general gene directed enzyme prodrug therapy (GDEPT) are related in that they also aim to destroy tumour cells by the tumour specific activation of a prodrug. However, in this case, the gene encoding the enzyme is either specifically targeted to malignant cells or is under the control of a specific promoter.
Up to now most of the efforts directed towards prodrug therapy have concentrated on the use of the human Herpes Simplex Virus thymidine kinase (HSV-tk) as a suicide gene. Although the HSV-tk enzyme in combination with the prodrug ganciclovir (GCV) has been recommended as a good system for GDEPT (Culver, K. et al., Science, 256:1550-1552 (1992); Ram, Z. et al., Cancer Research, 53:83-88 (1993); Chen, S. Shine, H. et al., Proc. Natl. Acad. Sci., 91:3054-3057 (1994)) there are a number of theoretical considerations that would suggest that it is by no means the best combination. First, it is an S-phase specific agent with no effect on resting cells. This is because the GCV monophosphate is short lived and has to be present when cells are entering the S-phase to give a toxic effect. The HSV-tk phosphorylates GCV to the monophosphate form (a reaction that cannot be performed by mammalian enzymes) which is then phosphorylated by cellular enzymes to the triphosphate form and incorporated into DNA. Second, the active drug is a triphosphate and would not be expected to diffuse freely to cause a bystander effect. However a bystander effect has been observed both in vitro and in vivo although metabolic cooperation appears to be involved and in the latter case some of the effect may be an indirect one involving an immune component (Bi, W., Parysek, L. et al., Human Gene Therapy, 4:725-731 (1993); Vile, R. and Hart, I., Cancer Research, 53:3860-3864 (1993) and Freeman, S., Abboud, C. et al., Cancer Research, 53:5274-5283 (1993)). One disadvantage is that the bystander effect is dependent on a cell-cell contact. This may be due to the presence of gap junctions formed by intimate contact between the transduced and the surrounding cells which enable the transfer of phosphorylated ganciclovir.
Recently, interesting results have been reported with cells that have been transfected with the gene encoding the rat cytochrome P450 form 2B1 and then treated with cyclophosphamide (Chen, S., Shine, H et al., Proc. Natl. Acad. Sci., 91:3054-3057 (1994)).
Cytochrome P450""s form a broad group of mono-oxygenases that catalyze oxidation of a wide range of substrates. They are produced by some bacteria, yeast, and by higher organisms, where they play a role in detoxification of xenobiotics, bioactivation reactions, and metabolism of various endogenous compounds.
Cytochrome P450 catalyzes the hydroxylation of the commonly used cancer prodrugs cyclophosphamide (CPA) and ifosfamide to their active toxic forms. Normally the expression of the patient""s endogenous cytochrome P450 gene is limited to the liver, and anti-tumour effects of systemically applied CPA depends upon the subsequent systemic distribution of toxic drug metabolites from the liver. This has led to toxicity problems since the activated drug not only affects the tumour but also affects other normal patient tissues such as bone marrow and kidney.
A therapeutic approach to overcome said systemic toxicity problems would be a direct delivery of the activated metabolites. Unfortunately said metabolites have after in vitro production a short half life of about 30 min. (Sladek, N. E., Powers, J. F. and Grage, G. M., Half-life of oxazaphosphorines in biological fluids. Drug Metab. Dispos., 12, 553-559 (1984)).
Thus, in an alternative therapeutic approach, as addressed in PCT/US95/10365, the cytochrome P450 gene is selectively introduced directly into tumour cells, and overexpressed in these cells. Toxic metabolites produced from the transduced cells affect surrounding non-transduced tumour cells in a concentration gradient dependent manner. An additional advantage of the cytochrome P-450/CPA system is the lack of dependency upon cell replication for cytotoxic effects on the surrounding cells. This is because one of the active metabolites generated causes interstrand crosslinks regardless of the cell cycle phase. Later on, during DNA synthesis, these interstrand crosslinks result in cell death.
For the treatment of cancers, it would be feasible to isolate cells from a patient (either tumour cells or normal cells) infect them in vitro with a recombinant retroviral particle carrying a gene encoding cytochrome P450, and then return them to the patient in the vicinity of the tumour. However, this approach is extremely labor intensive because each patient cells must be isolated, cultured, transduced with the gene construct and successfully returned without infection by adventitious agents. The cost and time involved in such an approach limits its practical usefulness. Moreover, most tumours are not suitable for ex vivo gene therapy.
Ideally, the gene encoding cytochrome P450 should be introduced in vivo into the tumour cells, or into cells in the vicinity of the tumour. PCT/US95/10365 suggests an in vivo infection of such cells with isolated retroviral particles. Unfortunately, retroviral particles have a very short half life in vivo and additionally, said particles are very quickly cleared by the immune system. Thus, the infection efficiency in normal tumours and thereby the expression of the prodrug activating enzyme in tumour cells is very poor.
In a further set-up of PCT/US95/10365 cytochrome P450 producing retroviral packaging cells were injected into the brain to provide the retroviral particles and additionally the activating enzyme at the site of a tumour. Even if, using this approach, the amount of activating enzyme could be increased, compared to the efficiency of only an infection with retroviral particles, it nevertheless has the drawback that this approach is clearly limited to the brain, since only the less active immune system in the brain would tolerate the injection of packaging cells, which is derived from a different organism.
Thus, it would be highly desirable if an approach could be envisaged, where one type of cells is transfected with the gene encoding P450 or infected with a recombinant retroviral particle carrying a gene encoding P450, and then used for therapy of many different patients as well as for many different tumours. Such an approach is much more feasible, assuming that problems of immune rejection can be overcome without weakening the patients immune status.
It is thus an object of the present invention to provide means, which allow the release of cytochrome P450 for the treatment of cancer or any other relevant disease without incidence of inflammatory or any other immune responses.
The invention inter alia comprises the following, alone or in combination:
A capsule encapsulating a cytochrome P450 producing cell, said capsule comprising a porous membrane which is permeable to cytochrome P450 produced by said cell;
the capsule as above, wherein the capsule material comprises a complex formed from cellulose sulphate and polydimethyldiallylammonium;
the capsule as any above, wherein the cytochrome P450 producing cell is a packaging cell line comprising a retroviral vector carrying the cytochrome P450 gene, said packaging cell line habouring at least one retroviral or recombinant retroviral construct coding for the proteins required for said retroviral vector to be packaged;
the capsule as above, wherein the retroviral vector is replication-defective;
the capsule as any above, wherein the retroviral vector comprises a 5xe2x80x2LTR region of the structure U3-R-U5; one or more sequences selected from coding and non-coding sequences wherein at least one of the coding sequences codes for cytochrome P450; and a 3xe2x80x2LTR region comprising a completely or partially deleted U3 region wherein said deleted U3 region is replaced by a polylinker sequence, followed by the R and U5 region;
the capsule as any above, wherein the cytochrome P450 gene is under transcriptional control of a target cell specific regulatory element or promoter, and/or an X-ray inducible promoter;
the capsule as any above, wherein the retroviral vector is a vector prepared as described in Example 2;
the capsule as above, wherein said cytochrome P450 producing cell comprises a vector prepared as described in Example 1;
the capsules as any above, wherein the encapsulated cells produce 0.1 to 10 pmol cytochrome P450 per 105 cells;
a pharmaceutical composition comprising the capsule as any above;
the capsule as any above for use in the treatment of cancer or any other relevant disease or disorder;
use of the capsule as any above for producing a pharmaceutical composition useful for the ablation of tumour cells;
a method of treating cancer or any other relevant disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of the capsule as any above and, either simultaneously or with a time span, a prodrug which is activated by cytochrome P450;
the method as above, wherein the prodrug is provided as a slow release preparation;
the method as any above, wherein the prodrug is encapsulated into a capsule comprising a porous membrane;
the method as any above, wherein the capsule is administered by injection and/or by implantion into the target organ and/or close to the site of said target organ, and the prodrug is administered systemically and/or locally;
the method as any above, wherein the capsule is administered by intra-arterial injection;
the method as any above, wherein the target organ comprises cells of breast tumours and/or pancreatic tumours;
the method as any above, wherein the prodrug is cyclophosphamide and/or ifosfamide;
the method as any above, wherein 10 to 100 mg per m2 body surface of said prodrug are administered to a subject in need thereof.
To achieve the foregoing and other objects, the present invention provides a capsule encapsulating cytochrome P450 producing cells, said capsule comprising a porous membrane which is permeable to cytochrome P450 produced by said cells. This capsule can be administered to a patient to increase at a desired location the amount of the liver enzyme cytochrome P450. Thus, the encapsulated cells injected or implanted into an individual provide a small prodrug conversion factory that can be near or in the tumour mass. According to one embodiment of the present invention the encapsulated cells produce about 0.1 to about 10 pmol cytochrome P450 per 105 cells. The cytochrome P450 released from said capsules in combination with a systemic or local application of ifosfamide or cyclophosphamide leads to high local concentrations of the activated metabolites, since cytochrome P450 hydroxylates said commonly used cancer prodrug to their toxic metabolites. This high local concentrations of said toxic metabolites affects surrounding tumour cells in a concentration gradient dependent manner without further systemic toxicity for the patient. Thus, the present invention provides novel means for an effective and well tolerable treatment of cancer or any other proliferative disease or disorder.
In one embodiment of the present invention the capsules encapsulate retroviral packaging cells which comprise a retroviral vector carrying the cytochrome P450 gene. Additionally, said packaging cells incorporates DNA which encodes for all proteins required for a retroviral particle to be packaged. Accordingly, said packaging cells produce the cytochrome P450 and additionally release retroviral particles, which themselves can transduce further cells by infection and subsequent integration of their vector genome carrying the cytochrome P450 gene into the genome of the infected cells. Thus, the encapsulated packaging cells producing said retroviral particles constitute a small virus producing factory, which can be placed at the site of application. This will allow efficient long term delivery of the recombinant virus in vivo.
The long term effectivity of this approach depends on (1) protection of the cells from the host immune system, which would normally eliminate virus producing or infected cells, especially if the cells are from a different species as is usually the case for retroviral vector producing cells and (2) survival of the cells in situ for extended periods, which may require vascularisation.
It has been found that the continuous production of a vector virus from implanted packaging cells can be achieved by the appropriate encapsulation, in microcapsules with semipermeable membranes, of the virus producing packaging cells before implantation. Additionally, it has been found that such capsules become well engrafted in the host, become vascularized, and do not elicit a host immune or inflammatory response. These findings, together with the semipermeability of the capsule membrane, permits long term retroviral vector delivery in vivo.
The pores in the porous membrane of the capsules according to the present invention have a size that allows cytochrome P450, any nutrition factors or viral particles to penetrate, but is not permeable for any cell of the immune system. Thus, cytochrome P450 producing cells inside the capsules are completely protected from cells of the host immune system and thus, induce no immune reaction even if said cells are allogenic or xenogenic.
The term xe2x80x9callogenicxe2x80x9d describes genetic differences within species, that is e.g. differences in cell surface markers such as MHC molecules on lymphocytes from genetically non-identical individuals. Allogenic cells are therefore simply those from another individual of the same species. In contrast thereto, the term xe2x80x9cxenogenicxe2x80x9d describes genetic differences between different species. Consequently, xenogenic cells are those from every other individual except representatives of the same species.
Due to said immune protection by the capsules the present invention therefore provides the possibility to encapsulate any allogenic or xenogenic cells that are transfected with an expression vector encoding the cytochrome P450 gene. In the typical expression vector according to the present invention the cytochrome P450 gene is under the transcriptional control of a strong constitutive promoter, such as the CMV promoter. Alternatively, an inducible or a X-ray dependent promoter can be used for expression of the cytochrome P450 gene. Said expression vectors are transfected into cells, according to standard protocols and subsequently, populations or clones of transduced cells, which produce about 0.1 pmol to about 10 pmol of cytochrome P450 are selected, characterized and encapsulated.
An encapsulation technology providing for the encapsulation of virus producing packaging cells, and of virus infected or normal cells in a non immunogenic, specifically in a cellulose based material, has been developed. Using this technique up to 1010, but preferably 105-107 cells are encapsulated in electrolyte complex (e.g. from alginate and polylysine or, more preferably, cellulose sulphate and polydimethyldiallylammonium chloride) or other porous structures (such as polyamides, polysulfones). The resulting capsules have a variable diameter between 0.01 and 5 mm, but preferably 0.1 and 1 mm. Consequently, capsules can be made to contain a variable number of cells. The capsule is semipermeable with pores that are large enough to allow viruses or prodrug molecules to pass through but small enough to prevent cells of the immune system from accessing the cells thereby significantly reducing an immune response to these cells. The capsules and the encapsulated cells are cultivated in a normal cell culture medium (the nature of which depends on the cell line encapsulated) at standard conditions of humidity, temperature and CO2 concentration.
After a suitable period in culture (normally not less than 1 hour and not exceeding 30 days), the cell containing capsules can be surgically implanted either directly, or by injection using a syringe into various areas.
At different times after the implantation of the encapsulated cells, the host can be treated with cyclosphosphamide or ifosfamide either locally or systemically. Cells infected with the cytochrome P450 expressing virus will convert these prodrugs to the active metabolites which cause alkylation and cross-linkage of DNA. Also cells carrying and expressing the cytochrome P450 gene (such as encapsulated infected cells, or encapsulated packaging cells) will also catalyze this conversion. In one embodiment of this invention these encapsulated infected or packaging cells will be either slowly dividing cells, or cells that have been treated with mitomycin C, low doses of radiation, or other means to prevent cell replication, and thus to prevent the cells from being themselves affected by the cytotoxic effects of the prodrugs.
For safety considerations a replication defective retroviral vector, in which the genes encoding for viral proteins have been replaced by heterologous DNA sequences is used. According to still a further embodiment of the present invention said vector comprises a 5xe2x80x2LTR region of the structure U3-R-U5, one or more sequences selected from coding and non-coding sequences wherein at least one of the coding sequences codes for cytochrome P450 and a 3xe2x80x2LTR region comprising a completely or partially deleted U3 region. Said deleted U3 region is replaced by a polylinker sequence followed by the R and U5 region. In still a further embodiment said polylinker is used to introduce a heterologous promoter, a target cell specific promoter and/or regulatory element or an X-ray inducible promoter into the 3xe2x80x2LTR of the retroviral vector. After infection of a new host cell said 3xe2x80x2LTR with the heterologous promoter element becomes duplicated and translocated to the 5xe2x80x2LTR and subsequently, controls the expression of the retroviral vector genome including the cytochrome P450 gene inserted into the body of the retroviral vector.
Accordingly, said vector does not undergo self-inactivation but instead promoter exchange, giving rise to the name ProCon vector for Promoter Conversion vectors. The principles and advantages of the ProCon system are described in more detail in WO 96/07748. For a complete disclosure of the present invention the disclosure of WO 96/07748 is incorporated herein.
Since Promoter Conversion does not result in Self-Inactivation, the retroviral vector will be transcriptionally active in the target cell. Additionally both LTR""s will consist to a large extent of heterologous promoter/enhancer sequences in the target cell. This will reduce the likelihood of the integrated vector in the target cell being subject to the same inactivation over long periods as has been described for conventional vectors (Xu, L., Yee, J. K. et al., Virology, 171:331-341 (1989)) and also will reduce the chance of recombination with endogenous retroviral sequences to generate potentially pathogenic replication competent virus, increasing the safety of the system.
According to the invention the 5xe2x80x2LTR of the retroviral vector construct is not modified, and expression of the viral vector in the packaging cells is driven by the normal retroviral U3 promoter. Normal retroviral polyadenylation is allowed, and no heterologous polyadenylation signals are included in the 3xe2x80x2LTR. This is important for the development of in vivo gene therapy strategies, since the normal physiological regulation of the virus, through the normal viral promoter, and possibly also involving the normal viral control of polyadenylation, will prevail over long periods in vivo whilst the packaging cells are producing recombinant virus.
The LTR regions used for a ProCon vector can be selected from at least one element of the group consisting of LTR""s of Murine Leukemia Virus (MLV), Mouse Mammary Tumour Virus (MMTV), Murine Sarcoma Virus (MSV), Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus (HIV), Human T-cell Leukemia Virus (HTLV), Feline Immunodeficiency Virus (FIV), Feline Leukemia Virus (FELV), Bovine Leukemia Virus (BLV) and Mason-Pfizer-Monkey virus (MPMV).
Alternatively, the retroviral vector is based on either a LXSN vector (Miller, A. D. and Rosman, G. J., Biotechniques, 7:980-990 (1989), PBAG (Price, J., Turner, D. et al., Proc. Natl. Acad. Sci. USA 84:156-160 (1987)) or a hybrid of both. The coding sequence of the therapeutic gene may be any cytochrome P450 gene but most preferably it is the rat cytochrome P450 form 2B1 defined by Fuji-Kuriyama, Y., Mizukami, Y., et al., Proc. Natl. Acad. Sci. USA 79:2793-2797 (1982)).
The promoters inserted into the 3xe2x80x2LTR can either be constitutive promoters such as the Cytomegalovirus (CMV) immediate early promoter/enhancer, inducible promoters such as promoters induced by glucocorticoid hormones (e.g., the MMTV promoter) or target cell specific promoters.
Such target cell specific regulatory elements and/or promoters are selected from one or more elements of any gene but preferably from promoters-such as carbonic anhydrase II, xcex2-glucokinase regulatory elements and/or promoters, lymphocyte specific regulatory elements and/or promoters, Whey Acidic Protein (WAP) elements and/or promoters, Mouse Mammary Tumour Virus (MMTV) elements and/or promoters, xcex2-lactoglobulin or casein specific regulatory elements and/or promoters, pancreas specific regulatory elements and/or promoters, immunoglobulin elements and/or promoters, MMTV lymphocytic specific regulatory elements and/or promoters, and/or MMTV specific regulatory elements and/or promoters conferring responsiveness to glucocorticoid hormones or directing expression to the mammary gland. Other promoters include for example the CD4, CD34, and IL2 promoters. Said regulatory elements and/or promoters regulate preferably the expression of said retroviral vector.
It appears that the region of the WAP promoter which is required for mediating the mammary gland specificity is a 320 bp XhoI/Xbal restriction fragment (xe2x88x92413 to xe2x88x9293) (Kolb, A. F., Gxc3xcnzburg, W. H., Albang, R., Brem, G., Erfle, V., and Salmons, B., Biochem. Biophys. Res. Commun., 217, 1045-1052 (1995)). In addition certain experiments indicate that a 0.6 Kb PstI MMTV promoter fragment (Salmons, B., Groner, B., Calberg Baca, C. M., and Ponta, H., Virology, 144:101-114 (1985)) may play a role in regulating the mammary gland specificity of expression displayed by MMTV (Kolb, A. F., Gxc3xcnzburg, W. H., Albang, R., Brem, G., Erfle, V., and Salmons, B., Biochem. Biophys. Res. Commun. 217, 1045-1052 (1995)).
According to standard protocols said retroviral vector is transfected into a packaging cell for the production of retroviral particles. Said packaging cells harbour at least one retroviral or recombinant retroviral construct coding for proteins required for said retroviral vector to be packaged. The packaging cells are either prepared from rodent, canine, feline or human cells or are chosen from one of the following cell lines: psi-2, psi-Crypt, psi-AM, GP+E-86, PA317 or GP+envAM-12, or of any of these supertransfected with recombinant constructs allowing expression of surface proteins from other enveloped viruses.
In the packaging cell line the expression of the retroviral vector is regulated by the normal unselective retroviral promoter contained in the U3 region of the 5xe2x80x2LTR. Accordingly, the packaging cell line and or the vector system is used to generate recombinant viruses that can be used to infect tumour or normal cells either in vitro or in vivo.
As soon as the recombinant virus infects the target cell promoter conversion occurs, and the P450 gene is expressed from a tissue specific or inducible promoter of choice inserted into the ProCon vector. Not only can virtually any tissue specific promoter be included in the system, providing for the selective targeting of a wide variety of different cell types, but additionally, following the conversion event, the structure and properties of the retroviral vector no longer resembles that of a virus. This, of course, has extremely important consequences from a safety point of view.
Recombinant retroviruses which have been purified or concentrated may be preserved by first adding a sufficient amount of a formulation buffer to the media containing the recombinant retrovirus, in order to form an aqueous suspension. The formulation buffer is an aqueous solution that contains a saccharide, a high molecular weight structural additive, and a buffering component in water. The aqueous solution may also contain one or more amino acids.
The recombinant retrovirus can also be preserved in a purified form. More specifically, prior to the addition of the formulation buffer, the crude recombinant retrovirus described above may be clarified by passing it through a filter, and then concentrated, such as by a cross flow concentrating system (Filtron Technology Corp., Nortborough, Mass.). Within one embodiment, DNase is added to the concentrate to digest exogenous DNA. The digest is then diafiltrated to remove excess media components and establish the recombinant retrovirus in a more desirable buffered solution. The diafiltrate is then passed over a Sephadex S-500 gel column and a purified recombinant retrovirus is eluted. A sufficient amount of formulation buffer is added to this eluate to reach a desired final concentration of the constituents and to minimally dilute the recombinant retrovirus, and the aqueous suspension is then stored, preferably at xe2x88x9270xc2x0 C. or immediately dried. As noted above, the formulation buffer is an aqueous solution that contains a saccharide, a high molecular weight structural additive, and a buffering component in water. The aqueous solution may also contain one or more amino acids.
The crude recombinant retrovirus can also be purified by ion exchange column chromatography. In general, the crude recombinant retrovirus is clarified by passing it through a filter, and the filtrate loaded onto a column containing a highly sulfonated cellulose matrix. The recombinant retrovirus is eluted from the column in purified form by using a high salt buffer. The high salt buffer is then exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column. A sufficient amount of formulation buffer is then added, as discussed above, to the purified recombinant retrovirus and the aqueous suspension is either dried immediately or stored, preferably at xe2x88x9270xc2x0 C.
The aqueous suspension in crude or purified form can be dried by lyophilisation or evaporation at ambient temperature. Specifically, lyophilisation involves the steps of cooling the aqueous suspension below the glass transition temperature or below the eutectic point temperature of the aqueous suspension, and removing water from the cooled suspension by sublimation to form a lyophilized retrovirus. Once lyophilized, the recombinant retrovirus is stable and may be stored at xe2x88x9220xc2x0 C. to 25xc2x0 C., as discussed in more detail below.
Within the evaporative method, water is removed from the aqueous suspension at ambient temperature by evaporation. Water can also be removed through spray drying.
The aqueous solutions used for formulation, as previously described, are composed of a saccharide, high molecular weight structural additive, a buffering component, and water. The solution may also include one or more amino acids. The combination of these components act to preserve the activity of the recombinant retrovirus upon freezing and lyophilisation, or drying through evaporation.
The high molecular weight structural additive aids in preventing viral aggregation during freezing and provides structural support in the lyophilized or dried state. Within the context of the present invention, structural additives are considered to be of xe2x80x9chigh molecular weight (MW)xe2x80x9d if they are greater than 5000 MW. A preferred high molecular weight structural additive is human serum albumin.
The amino acids, if present, function to further preserve viral infectivity upon cooling and thawing of the aqueous suspension. In addition, amino acids function to further preserve viral infectivity during sublimation of the cooled aqueous suspension and while in the lyophilized state.
The buffering component acts to buffer the solution by maintaining a relatively constant pH. A variety of buffers may be used, depending on the pH range desired, preferably between 7.0 and 7.8.
Aqueous solutions for the formulation of recombinant retroviruses are described in detail in WO-A2-96121014.
In addition, it is preferable that the aqueous solution contain a neutral salt which is used to adjust the final formulated recombinant retrovirus to an appropriate isosmotic salt concentration.
Lyophilized or dehydrated retroviruses may be reconstituted using a variety of substances, but are preferably reconstituted using water. In certain instances, dilute salt solutions which bring the final formulation to isotonicity may also be used. In addition, it may be advantageous to use aqueous solutions containing components known to enhance the activity of the reconstituted retrovirus. Such components include cytokines, such as IL-2, polycations, such as prolamine sulfate, or other components which enhance the transduction efficiency of the reconstituted retrovirus. Lyophilized or dehydrated recombinant retrovirus may be reconstituted with any convenient volume of water or the reconstituting agents that allow substantial, and preferably total, solubilization of the lyophilized or dehydrated sample.
Recombinant retroviral particles may be administered to a wide variety of locations including, for example, into sites such as an organ or to a site of a tumour. Within other embodiments, the recombinant retrovirus may be administered orally, intravenously, buccal/sublingual, intraperitoneally, or subcutaneously. The daily dosage depends upon the exact mode of administration, form in which administered, the indication toward which the administration is directed, the subject involved and the body weight of the subject involved, and further the preference and experience of the physician in charge.
The routes of administration described herein may be accomplished simply by direct administration using a needle, catheter or related device. In particular, within certain embodiments of the invention, one or more dosages may be administered directly.
The present invention also provides a pharmaceutical composition wherein the capsules encapsulating cytochrome P450 producing cells carrying the cytochrome P450 gene are mixed with a pharmaceutical acceptable carrier or diluent.
According to a further embodiment the capsules encapsulating cytochrome P450 producing cells and/or said pharmaceutical composition is used for the treatment of cancer or any other relevant disease or disorder. For an effective treatment of cancer according to the present invention a method comprising in addition to the implantation or injection of the capsules a systemic and/or local administration of the activatable prodrug is provided. This administration can be performed simultaneously together with the implantation or injection of the capsules. However, an administration of the prodrug with a time delay is possible as well. In a further embodiment of the present invention the prodrug is provided as a slow release preparation and/or encapsulated in a capsule comprising a porous membrane. This slow release preparation and also the encapsulated prodrug prolong the period, wherein prodrug is released at the site of application and thus, the cytochrome P450 catalyzes activation of toxic metabolites at said site of application.
According to the present invention the capsules are administered by injection or implantation into the target organ or close to said target organ. It proofed as advantageous according to still a further embodiment to inject the capsules directly into the artery which supplies the target organ to be treated, thus the capsules get flushed directly into the target organ. This is especially useful for the treatment of human pancreatic adenocarcinoma, since this tumour is not very suitable for resection. Additionally, this intra-arterial injection is also useful for any other tumour in organs of glandular tissue such as the breast, the kidney or the prostate gland.
In a further preferred embodiment ifosfamide or cyclophosphamide are used as prodrug. Furthermore, a therapeutic amount of 10 to 100 mg/m2 body surface of said prodrug is applied to a patient in need thereof.
The detailed Examples which follow are intended to contribute to a better understanding of the present invention. However, it is not intended to give the impression that the invention is confined to the subject-matter of the Examples.